Obesity is a major health problem that predisposes people to diabetes, cancer, and heart disease. Despite considerable effort, the precise molecular mechanisms that regulate body weight are not well characterized. Neuropeptides are thought to play a critical role in regulating feeding, but these molecules are often missed by proteomics techniques due to their small size and low abundance. We have recently developed a method to isolate neuropeptide-processing intermediates from fat/fat mouse brain, and have used differential isotopic labeling to provide relative quantitation of peptides from two different animals (or pools of animals). Using this quantitative neuropeptidomics method, we have detected a large number of peptides in mouse hypothalamus, of which 20-30% are substantially up- or down-regulated by 2 days of food deprivation. In Aim 1, we will identify these peptides using tandem mass spectrometry. At the same time, in Aim 2 we will develop additional isotopic labels that allow for the quantitation of a larger number of peptides, and which provide more information from each experiment. Aim 3 will use either the new labels developed in Aim 2 (if successful) or the existing labels currently in use to examine additional paradigms of food deprivation and re-feeding. It is anticipated that these studies will lead to the identification of novel neuropeptides that are altered by food deprivation and/or re-feeding after fasting. In further studies, it would be important to test if these peptides are similarly regulated in wild type mice, and whether microinjection of the peptides influence feeding and/or body weight; these additional studies are beyond the scope of this R21 application, but would be continued in a subsequent R01 application. The studies in this initial R21 will provide an unbiased survey of neuropeptides that are altered by food deprivation, and which may therefore be involved in regulating food intake and/or body weight.